New Inv. Award: Developing single-cell isoform sequencing tools to explore the diversity and regulation of alternative splicing in haematopoiesis

细胞是生物学的基本单位——所有多细胞生物都由数十亿甚至数万亿个细胞组成——其中许多细胞在生物体内具有不同的功能。细胞功能的多样性源于细胞调节和编排这些细胞表达的基因的能力。细胞用来增加这一序列复杂性的一个关键机制是一个被称为选择性剪接的过程。这是一个受调控的过程，在基因表达过程中，遗传信息可以选择性地从信使RNA分子中切除。这可能导致单个基因产生多种蛋白质变体，并且这些变体通常在细胞中具有不同的功能角色。正是通过这种方法，人类基因组中超过20,000个蛋白质编码基因的功能复杂性可以增加5-10倍-因此，从相对较少的基因中，可以获得更多种类的基因表达和功能。DNA测序技术的最新进展使研究人员能够研究单细胞中包含的遗传信息——RNA、DNA和DNA的表观遗传修饰。这让我们对构成生物体的细胞类型的复杂性和多样性有了全新的认识。这些技术广泛适用于不同的生物体，也适用于人类健康和疾病。然而，迄今为止，尽管这一过程在植物、动物和人类的正常发育以及癌症等人类疾病中发挥着重要作用，但对单细胞中选择性剪接的性质探索甚少。在这项提议中，我们寻求产生新的方法，不仅揭示单细胞和小细胞群体中选择性剪接的程度，而且对这一过程的调节提供平行的见解。虽然我们开发的方法可以应用于几乎任何多细胞生物，但我们将使用该技术探索小鼠正常血细胞发育的这些过程。我们之前已经开发了对单细胞基因组和转录组进行平行分析的方法，并进一步开发了这些方法以包括表观遗传信息-以DNA甲基化的形式。通过将这些方法扩展到所谓的“长读”测序技术，我们将创建一个平台，使我们能够读取单个细胞中剪接变异的全部补充。与此同时，我们将能够探索DNA甲基化如何调节选择性剪接。通过使用正常血细胞发育作为这项新技术的试验场，我们将首次揭示小细胞群体和单细胞中选择性剪接的变异量，其功能已被很好地理解。这些信息将有助于增强我们对血液干细胞如何做出决定的理解，以及这个复杂的系统如何维持每天数十亿个新细胞的产生。此外，通过观察来自年轻和老年小鼠的细胞，我们将研究这些细胞中选择性剪接的使用如何随着年龄的变化而变化。

The cell is a fundamental unit of biology - all multicellular organisms consist of populations of billions, even trillions of cells - many of which will have differing functions within the organism. The diversity of cell function arises from the ability of the cell to regulate and orchestrate the repertoire of genes those cells express. One key mechanism cells use to increase the complexity of this repertoire is a process called alternative splicing. This is a regulated process where, during the process of gene expression, genetic information can be selectively excised from messenger RNA molecules. This can result in the generation of multiple protein variants from a single gene, and often these variants can have functionally distinct roles in the cell. It is by this means that the functional complexity of over 20,000 protein coding genes in the human genome can be increased by a factor of 5-10 - so from a relatively small number of genes, a larger variety of gene expression and function is possible. Recent advances in DNA sequencing technology have enabled researchers to study the genetic information - RNA, DNA and epigenetic modifications to the DNA - contained within single cells. This has allowed a totally new perspective on the complexity and diversity of cell types that make up an organism. These techniques are broadly applicable to different organisms, and in human health and disease. However, to date, little has been done to explore the nature of alternative splicing in single cells, in spite of the important role this process plays in normal development of plants, animals and humans, and indeed in human diseases such as cancer.In this proposal, we seek to generate new approaches that will reveal not just the extent of alternative splicing in single cells, and small populations of cells, but give parallel insight into the regulation of this process. While the methods we develop could be applied to almost any multicellular organism, we will use the technique to explore these processes in the development of normal blood cells in the mouse. We have previously developed methods for parallel analysis of the genomes and transcriptomes of single cells, and further developed these methods to include epigenetic information - in the form of DNA methylation. By expanding these methods to work with so-called "long read" sequencing technology, we will create a platform which allows us to read out the full complement of splicing variation in individual cells. In parallel we will be able to explore how alternative splicing might be regulated by DNA methylation.By using normal blood cell development as a testing ground for this new technology, we will reveal for the first time the amount of variation in alternative splicing in small populations of cells and single cells for which the function is very well understood. This information will be useful in enhancing our understanding of how blood stem cells make decisions, and how this complex system can sustain the generation of billions of new cells every day. Furthermore, by looking at cells from young and aged mice we will examine how the use of alternative splicing changes with age in these cells.